2&#39;-fluoropyrimidine anti-calf intestinal phosphatase nucleic acid ligands

ABSTRACT

Methods are described for the identification and preparation of nucleic acid ligands to calf intestinal phosphatase. Included in the invention are specific RNA ligands to calf intestinal phosphatase identified by the SELEX method.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.10/096,830, filed Mar. 12, 2002, which is a continuation of U.S.application Ser. No. 09/941,964, filed Aug. 28, 2001, now U.S. Pat. No.6,387,635, which is a divisional of U.S. application Ser. No.09/335,012, filed Jun. 17, 1999, now U.S. Pat. No. 6,280,943, each ofwhich is entitled “2′-Fluoropyrimidine Anti-Calf Intestinal PhosphataseNucleic Acid Ligands.”

FIELD OF THE INVENTION

[0002] Described herein are high affinity nucleic acid ligands to calfintestinal phosphatase (CIP). Also described herein are methods foridentifying and preparing high affinity nucleic acid ligands to CIP. Themethod used herein for identifying such nucleic acid ligands is calledSELEX, an acronym for Systematic Evolution of Ligands by Exponentialenrichment. Further disclosed are RNA ligands to CIP. Also included areoligonucleotides containing nucleotide derivatives chemically modifiedat the 2′-positions of pyrimidines. Additionally disclosed are RNAligands to CIP containing 2′-F modifications. The invention alsoincludes high affinity nucleic acid inhibitors of CIP. Theoligonucleotides of the present invention are useful as diagnosticagents.

BACKGROUND OF THE INVENTION

[0003] Calf intestinal alkaline phosphatase (CIP) is a commonly usedreporter enzyme for research and clinical assays. These assays typicallyuse synthetic substrates that become detectable upon removal ofphosphate groups by CIP. For example, one commonly used substrate is 1,2dioxetane. This substrate becomes chemiluminescent upon the removal ofphosphate groups by CIP. Another common substrate is the chromagenp-nitrophenylphosphate. Antibodies conjugated to CIP are widely used inELISA, and nucleic acid probes linked to CIP can be used in a variety ofnucleic acid detection schemes. Given the widespread use of CIP invarious target detection schemes, it would be desirable to provide aclass of ligands distinct from antibodies to which CIP could beconjugated.

[0004] The dogma for many years was that nucleic acids had primarily aninformational role. Through a method known as Systematic Evolution ofLigands by EXponential enrichment, termed the SELEX process, it hasbecome clear that nucleic acids have three dimensional structuraldiversity not unlike proteins. The SELEX process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment,” now abandoned, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands,” and U.S. Pat. No. 5,270,163(see also WO 91/19813), entitled “Methods for Identifying Nucleic AcidLigands,” each of which is specifically incorporated by reference hereinin its entirety. Each of these applications, collectively referred toherein as the SELEX Patent Applications, describes a fundamentally novelmethod for making a nucleic acid ligand to any desired target molecule.

[0005] The SELEX process provides a class of products that are referredto as nucleic acid ligands or aptamers, each having a unique sequence,and which have the property of binding specifically to a desired targetcompound or molecule. Each SELEX-identified nucleic acid ligand isa-specific ligand of a given target compound or molecule. The SELEXprocess is based on the unique insight that nucleic acids havesufficient capacity for forming a variety of two- and three-dimensionalstructures and sufficient chemical versatility available within theirmonomers to act as ligands (form specific binding pairs) with virtuallyany chemical compound, whether monomeric or polymeric. Molecules of anysize or composition can serve as targets in the SELEX method. The SELEXmethod applied to the application of high affinity binding involvesselection from a mixture of candidate oligonucleotides and step-wiseiterations of binding, partitioning and amplification, using the samegeneral selection scheme, to achieve virtually any desired criterion ofbinding affinity and selectivity. Starting from a mixture of nucleicacids, preferably comprising a segment of randomized sequence, the SELEXmethod includes steps of contacting the mixture with the target underconditions favorable for binding, partitioning unbound nucleic acidsfrom those nucleic acids which have bound specifically to targetmolecules, dissociating the nucleic acid-target complexes, amplifyingthe nucleic acids dissociated from the nucleic acid-target complexes toyield a ligand-enriched mixture of nucleic acids, then reiterating thesteps of binding, partitioning, dissociating and amplifying through asmany cycles as desired to yield highly specific high affinity nucleicacid ligands to the target molecule.

[0006] It has been recognized by the present inventors that the SELEXmethod demonstrates that nucleic acids as chemical compounds can form awide array of shapes, sizes and configurations, and are capable of a farbroader repertoire of binding and other functions than those displayedby nucleic acids in biological systems.

[0007] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” now abandoned (see, U.S. Pat. No. 5,707,796),describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. Pat.No. 6,001,577, entitled “Systematic Evolution of Nucleic Acid Ligands byExponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” describe a SELEX based method for selecting nucleicacid ligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, which can be non-peptidic, termedCounter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolutionof Ligands by EXponential Enrichment: Solution SELEX,” describes aSELEX-based method which achieves highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.

[0008] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,580,737, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known & Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

[0009] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459, entitled “SystematicEvolution of Ligands by EXponential Enrichment: Chimeric SELEX,” andU.S. Pat. No. 5,683,867 entitled “Systematic Evolution of Ligands byEXponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0010] The SELEX method further encompasses combining selected nucleicacid ligands with lipophilic compounds or non-immunogenic, highmolecular weight compounds in a diagnostic or therapeutic complex asdescribed in U.S. Pat. No. 6,011,020, entitled “Nucleic Acid Complexes.”Each of the above described patents and applications which describemodifications of the basic SELEX procedure are specifically incorporatedby reference herein in their entirety.

[0011] It is an object of the present invention to provide methods thatcan be used to identify nucleic acid ligands that bind with highspecificity and affinity to calf intestinal phosphatase (CIP).

[0012] It is a further object of the present invention to obtain nucleicacid ligands to CIP that inhibit the activity of CIP when bound.

[0013] It is also an object of the present invention to obtain nucleicacid ligands to CIP that do not inhibit the phosphatase activity whenbound to CIP.

[0014] An even further object of the invention is to provide a methodfor performing SELEX in a robotics-compatible microtiter plate format.

[0015] A still further object of the invention is to provide a methodfor absorbing SELEX targets to solid support surfaces, includingmicrotiter plates, solely through hydrophobic interactions.

SUMMARY OF THE INVENTION

[0016] The present invention describes a method for isolating nucleicacid ligands that bind to calf intestinal phosphatase (CIP) with highspecificity. The nucleic acid ligands of the invention can either beinhibitory or non-inhibitory. High affinity anti-CIP nucleic acidligands can have many potential uses in assay systems that use CIP as areporter enzyme.

[0017] The present invention also provides methods for immobilizing aSELEX target on a robotics-compatible microtiter plate solely throughhydrophobic interactions. This method will allow high-throughputautomation of the SELEX process.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGS. 1A-D illustrate nucleic acid ligand sequences obtained fromthe round 8 SELEX pool.

[0019]FIG. 2 illustrates CIP binding isotherms for the determination ofthe equilibrium dissociation constant at 37° C. for random 2′-F RNA(black squares) and the round 8 SELEX pool (black circles). Data areshown as the mean+/− the SEM (standard error margins) for duplicatedeterminations at each point.

[0020]FIG. 3 depicts CIP binding isotherms for the determination of theequilibrium dissociation constant at 37° C. for individual clones fromthe round 8 SELEX pool. Data are shown as the mean+/− the SEM forduplicate determinations at each point.

[0021]FIG. 4 depicts CIP inhibition experiments. Random 2′-F RNA (R),along with the nucleic acid ligand sequences DD-172-2-4 (4) (SEQ IDNO:35), DD-172-2-9 (9) (SEQ ID NO:8), DD-172-2-17 (17) (SEQ ID NO:2),DD-172-2-20 (20) (SEQ ID NO:13) and DD-172-2-29 (29) (SEQ ID NO:3), weretested for their ability to inhibit the activity of CIP. Data are shownas the mean+/− the standard deviation for duplicate determinations. Datafrom each group were compared to CIP activity in the absence of addednucleic acid (0).

DETAILED DESCRIPTION OF THE INVENTION

[0022] The central method utilized herein for identifying nucleic acidligands to CIP is called the SELEX process, an acronym for SystematicEvolution of Ligands by Exponential enrichment and involves: a)contacting the candidate mixture of nucleic acids with calf intestinalphosphatase, or expressed domains or peptides corresponding to calfintestinal phosphatase, b) partitioning between members of saidcandidate mixture on the basis of affinity to calf intestinalphosphatase, and c) amplifying the selected molecules to yield a mixtureof nucleic acids enriched for nucleic acid sequences with a relativelyhigher affinity for binding to calf intestinal phosphatase.

[0023] Definitions

[0024] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided:

[0025] As used herein, “nucleic acid ligand” is a non-naturallyoccurring nucleic acid having a desirable action on a target. Nucleicacid ligands are often referred to as “aptamers”. A desirable actionincludes, but is not limited to, binding of the target, catalyticallychanging the target, reacting with the target in a way whichmodifies/alters the target or the functional activity of the target,covalently attaching to the target as in a suicide inhibitor,facilitating the reaction between the target and another molecule. Inthe preferred embodiment, the action is specific binding affinity for atarget molecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which predominantly depends on Watson/Crickbase pairing or triple helix binding, wherein the nucleic acid ligand isnot a nucleic acid having the known physiological function of beingbound by the target molecule. In the present invention, the target iscalf intestinal phosphatase, or regions thereof. Nucleic acid ligandsinclude nucleic acids that are identified from a candidate mixture ofnucleic acids, said nucleic acid ligand being a ligand of a giventarget, by the method comprising: a) contacting the candidate mixturewith the target, wherein nucleic acids having an increased affinity tothe target relative to the candidate mixture may be partitioned from theremainder of the candidate mixture; b) partitioning the increasedaffinity nucleic acids from the remainder of the candidate mixture; andc) amplifying the increased affinity nucleic acids to yield aligand-enriched mixture of nucleic acids.

[0026] As used herein, “candidate mixture” is a mixture of nucleic acidsof differing sequence from which to select a desired ligand. The sourceof a candidate mixture can be from naturally-occurring nucleic acids orfragments thereof, chemically synthesized nucleic acids, enzymaticallysynthesized nucleic acids or nucleic acids made by a combination of theforegoing techniques. In a preferred embodiment, each nucleic acid hasfixed sequences surrounding a randomized region to facilitate theamplification process.

[0027] As used herein, “nucleic acid” means either DNA, RNA,single-stranded or double-stranded, and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

[0028] “SELEX” methodology involves the combination of selection ofnucleic acid ligands which interact with a target in a desirable manner,for example binding to a protein, with amplification of those selectednucleic acids. Optional iterative cycling of the selection/amplificationsteps allows selection of one or a small number of nucleic acids whichinteract most strongly with the target from a pool which contains a verylarge number of nucleic acids. Cycling of the selection/amplificationprocedure is continued until a selected goal is achieved. In the presentinvention, the SELEX methodology is employed to obtain nucleic acidligands to calf intestinal phosphatase.

[0029] The SELEX methodology is described in the SELEX PatentApplications.

[0030] “SELEX target” or “target” means any compound or molecule ofinterest for which a ligand is desired. A target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.without limitation. In this application, the SELEX target is calfintestinal phosphatase. In particular, the SELEX targets in thisapplication include purified calf intestinal phosphatase, and fragmentsthereof, and short peptides or expressed protein domains comprising calfintestinal phosphatase.

[0031] As used herein, “solid support” is defined as any surface towhich molecules may be attached through either covalent or non-covalentbonds. This includes, but is not limited to, membranes, microtiterplates, magnetic beads, charged paper, nylon, Langmuir-Bodgett films,functionalized glass, germanium, silicon, PTFE, polystyrene, galliumarsenide, gold, and silver. Any other material known in the art that iscapable of having functional groups such as amino, carboxyl, thiol orhydroxyl incorporated on its surface, is also contemplated. Thisincludes surfaces with any topology, including, but not limited to,spherical surfaces and grooved surfaces.

[0032] Preparation of Nucleic Acid Ligands to Calf IntestinalPhosphatase

[0033] In the preferred embodiment, the nucleic acid ligands of thepresent invention are derived from the SELEX methodology. The SELEXprocess is described in U.S. patent application Ser. No. 07/536,428,entitled “Systematic Evolution of Ligands by Exponential Enrichment,”now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands,”and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methodsfor Identifying Nucleic Acid Ligands.” These applications, eachspecifically incorporated herein by reference, are collectively calledthe SELEX Patent Applications.

[0034] The SELEX process provides a class of products which are nucleicacid molecules, each having a unique sequence, and each of which has theproperty of binding specifically to a desired target compound ormolecule. Target molecules are preferably proteins, but can also includeamong others carbohydrates, peptidoglycans and a variety of smallmolecules. SELEX methodology can also be used to target biologicalstructures, such as cell surfaces or viruses, through specificinteraction with a molecule that is an integral part of that biologicalstructure.

[0035] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0036] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: a) to assistin the amplification steps described below, b) to mimic a sequence knownto bind to the target, or c) to enhance the concentration of a givenstructural arrangement of the nucleic acids in the candidate mixture.The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0037] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0038] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0039] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity for the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0040] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer uniquesequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0041] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No.5,707,796, both entitled “Method for Selecting Nucleic Acids on theBasis of Structure,” now abandoned (see, U.S. Pat. No. 5,707,796),describe the use of the SELEX process in conjunction with gelelectrophoresis to select nucleic acid molecules with specificstructural characteristics, such as bent DNA. U.S. patent applicationSer. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection ofNucleic Acid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. Pat.No. 6,001,577, entitled “Systematic Evolution of Nucleic Acid Ligands byExponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” all describe a SELEX based method for selecting nucleicacid ligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands ThatDiscriminate Between Theophylline and Caffeine,” describes a method foridentifying highly specific nucleic acid ligands able to discriminatebetween closely related molecules, termed Counter-SELEX. U.S. Pat. No.5,567,588, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Solution SELEX,” describes a SELEX-based method whichachieves highly efficient partitioning between oligonucleotides havinghigh and low affinity for a target molecule. U.S. Pat. No. 5,496,938,entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev,” describesmethods for obtaining improved nucleic acid ligands after SELEX has beenperformed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution ofLigands by Exponential Enrichment: Chemi-SELEX,” describes methods forcovalently linking a ligand to its target.

[0042] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,”that describes oligonucleotides containing nucleotide derivativeschemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat.No. 5,637,459, supra, describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent applicationSer. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method ofPreparation of Known and Novel 2′ Modified Nucleosides by IntramolecularNucleophilic Displacement,” now abandoned, describes oligonucleotidescontaining various 2′-modified pyrimidines.

[0043] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. Pat. No. 5,637,459, entitled “SystematicEvolution of Ligands by Exponential Enrichment: Chimeric SELEX” and U.S.Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands byExponential Enrichment: Blended SELEX,” respectively. These applicationsallow the combination of the broad array of shapes and other properties,and the efficient amplification and replication properties, ofoligonucleotides with the desirable properties of other molecules.

[0044] In U.S. Pat. No. 5,496,938 methods are described for obtainingimproved nucleic acid ligands after the SELEX process has beenperformed. This patent, entitled “Nucleic Acid Ligands to HIV-RT andHIV-1 Rev,” is specifically incorporated herein by reference.

[0045] One potential problem encountered in the diagnostic use ofnucleic acids is that oligonucleotides in their phosphodiester form maybe quickly degraded in body fluids by intracellular and extracellularenzymes such as endonucleases and exonucleases before the desired effectis manifest. Certain chemical modifications of the nucleic acid ligandcan be made to increase the in vivo stability of the nucleic acid ligandor to enhance or to mediate the delivery of the nucleic acid ligand.See, e.g., U.S. patent application Ser. No. 08/117,991, filed Sep. 8,1993, now abandoned, and U.S. Pat. No. 5,660,985, both entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” whichare specifically incorporated herein by reference. Modifications of thenucleic acid ligands contemplated in this invention include, but are notlimited to, those which provide other chemical groups that incorporateadditional charge, polarizability, hydrophobicity, hydrogen bonding,electrostatic interaction, and fluxionality to the nucleic acid ligandbases or to the nucleic acid ligand as a whole. Such modificationsinclude, but are not limited to, 2′-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at exocyclic amines, substitution of 4-thiouridine,substitution of 5-bromo or 5-iodo-uracil; backbone modifications,phosphorothioate or alkyl phosphate modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping. In preferred embodiments of the instantinvention, the nucleic acid ligands are RNA molecules that are 2′-fluoro(2′-F) modified on the sugar moiety of pyrimidine residues.

[0046] The modifications can be pre- or post-SELEX processmodifications. Pre-SELEX process modifications yield nucleic acidligands with both specificity for their SELEX target and improved invivo stability. Post-SELEX process modifications made to 2′-OH nucleicacid ligands can result in improved in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

[0047] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX process (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process.

[0048] The nucleic acid ligands of the invention are prepared throughthe SELEX methodology that is outlined above and thoroughly enabled inthe SELEX applications incorporated herein by reference in theirentirety. The SELEX process can be performed using purified calfintestinal phosphatase, or fragments thereof as a target. Alternatively,full-length calf intestinal phosphatase, or discrete domains of calfintestinal phosphatase, can be produced in a suitable expression system.Alternatively, the SELEX process can be performed using as a target asynthetic peptide that includes sequences found in calf intestinalphosphatase. Determination of the precise number of amino acids neededfor the optimal nucleic acid ligand is routine experimentation forskilled artisans.

[0049] In some embodiments, the nucleic acid ligands become covalentlyattached to their targets upon irradiation of the nucleic acid ligandwith light having a selected wavelength. Methods for obtaining suchnucleic acid ligands are detailed in U.S. patent application Ser. No.08/123,935, filed Sep. 17, 1993, entitled “Photoselection of NucleicAcid Ligands,” now abandoned, U.S. Pat. No. 5,763,177, entitled“Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment:Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. Pat.No. 6,001,577, entitled “Systematic Evolution of Nucleic Acid Ligands byExponential Enrichment: Photoselection of Nucleic Acid Ligands andSolution SELEX,” each of which is specifically incorporated herein byreference in its entirety.

[0050] In preferred embodiments, the SELEX process is carried out usingfull length calf intestinal phosphatase coated on the surface of wellsof a plastic microtiter plate. A candidate mixture of single strandedRNA molecules is then contacted with the bound calf intestinalphosphatase in the wells of the plate. After incubation for apredetermined time at a selected temperature, the wells of the plate arewashed to remove unbound candidate nucleic acid ligand. The nucleic acidligand that binds to the calf intestinal phosphatase is then releasedinto solution, and amplified using the Polymerase Chain Reaction. Theamplified candidate mixture is then used to begin the next round of theSELEX process.

[0051] After nucleic acid ligands with the desired affinity for calfintestinal phosphatase are isolated, they can be assayed to determine ifthey inhibit the activity of the enzyme. This can be performed using anyof the numerous methods known in the art for the determination of calfintestinal phosphatase activity. For example, in some embodiments, theassay used can be the chromagenic p-Nitrophenylphosphate assay.

[0052] The nucleic acid ligands isolated by the method of the instantinvention have a great number of applications in assays systems that usecalf intestinal phosphatase. For example, in some embodiments, anon-inhibitory nucleic acid ligand is conjugated to calf intestinalphosphatase in such a way that the calf intestinal phosphatase does notbind to the affinity site on the nucleic acid ligand. The calfintestinal phosphatase molecule to which the nucleic acid ligand isconjugated can itself be conjugated to a target analyte specificreagent. When this analyte specific reagent binds to its target analyte,calf intestinal phosphatase can be added to the solution, and will bindto the nucleic acid ligand conjugated to the original molecule of calfintestinal phosphatase. This binding of additional calf intestinalphosphatase to the target analyte specific reagent will result in agreatly enhanced signal upon addition of the detectable substrate. Inthis way, the sensitivity of many assay systems that use analytespecific reagents conjugated to calf intestinal phosphatase—such as calfintestinal phosphatase conjugated antibodies—can be dramaticallyincreased.

[0053] In other embodiments, a nucleic acid molecule can be synthesizedin which the calf intestinal phosphatase nucleic acid ligand sequence iscontiguous with a nucleic acid ligand sequence directed against anothertarget molecule. The resulting nucleic acid ligand can bind to thetarget molecule, and this binding can then be detected by adding calfintestinal phosphatase and the appropriate substrate. In this way, theamount of target analyte present is directly proportional to the amountof signal generated by CIP activity on the substrate.

[0054] In other embodiments, non-inhibitory CIP nucleic acid ligands canbe conjugated to any other reagent that is capable of binding to aparticular analyte. For example, the nucleic acid ligand could beconjugated to antibodies, proteins, sugars or peptides. Such reagentsmay have longer shelf-lives than traditional antibody-conjugated calfintestinal phosphatase, as the actual enzyme is added separately fromthe ligand-conjugated detection reagent.

[0055] Inhibitory nucleic acid ligands of calf intestinal phosphatasealso have great utility. Inhibitory calf intestinal phosphatase nucleicacid ligands could be used in a homogeneous assay system for thedetection of proteins. In this embodiment, an inhibitory CIP nucleicacid ligand is synthesized contiguously with a nucleic acid ligandspecific for an independent target analyte such that the binding of CIPand the target analyte are mutually exclusive. The system is designedwith CIP, CIP substrate and the nucleic acid ligand present atconcentrations that nearly completely inhibit CIP activity. Addition ofthe test solution containing the specific analyte will result in therelease of CIP from the nucleic acid ligand. Thus increasingconcentrations of analyte will be directly proportional to the CIPsignal achieved. Inhibitory nucleic acid ligand can be used withoutmodification, but if necessary stronger CIP inhibitors can be created byconjugating a small molecule CIP inhibitor to the aptamer or by adding alarge molecular weight moiety such as polyethylene glycol, dextran, tohelp the inhibitory properties via steric hindrance. Naturally, any ofthese embodiments are not limited to CIP as the reporter enzyme, but areequally applicable to other enzyme reporters including, but not limitedto, enzymes such as peroxidase, beta-galactosidase, glucose-6-phosphatedehydrogenase, and glucose oxidase.

[0056] The SELEX target of the instant invention, CIP, can beimmobilized solely through hydrophobic interactions with the surface ofa microtiter plate during the SELEX process. This immobilization to asolid support allows the washing and elution steps to be carried out inthe microtiter plate. Previous embodiments of the SELEX process haveimmobilized targets to solid supports by modifying the target in someway. Such modifications require manipulation of the targets and areoften time consuming. By contrast, the hydrophobic immobilization methodprovided by the instant invention is extremely simple, requiring nomanipulation of the target. Moreover, the use of a robotics-compatiblemicrotiter plate format allows the SELEX process to be automated.

EXAMPLES

[0057] The following examples are given by way of illustration only.They are not to be taken as limiting the scope of the present invention.

Example 1 Use of SELEX to Obtain Nucleic Acid Ligands to Calf IntestinalPhosphatase

[0058] Calf intestinal alkaline phosphatase (10 mg/ml; molecular weight52,486 Daltons) was purchased from Boehringer Mannheim (Indianapolis,Ind.). Single-stranded DNA-primers and templates were synthesized byOperon Technologies Inc. (Alameda, Calif.).

[0059] The SELEX-process has been described in detail in: Fitzwater andPolisky (1996) Methods Enzymol. 267:275-301. In brief, double strandedtranscription templates were prepared by Klenow fragment extension of40N8 ssDNA: 5′-gcctgttgtgagcctcctgtcgaa(n₄₀)ttgagcgtttattcttgt ctccc3′(SEQ ID NO:44) using the 5N8 primer:5′-taatacgactcactatagggagacaagaataaacgctcaa-3′ (SEQ ID NO:45) thatcontains the T7 polymerase promoter (underlined). RNA was prepared withT7 RNA polymerase as described previously in Fitzwater and Polisky(1996) Methods Enzymol. 267:275-301, incorporated herein by reference inits entirety. All transcription reactions were performed in the presenceof pyrimidine nucleotides that were 2′-fluoro (2′-F) modified on thesugar moiety. This substitution confers enhanced resistance toribonucleases that utilize the 2′-hydroxyl moiety for cleavage of thephosphodiester bond. Specifically, each transcription mixture contained3.3 mM 2′-F UTP and 3.3 mM 2′-F CTP along with 1 mM GTP and ATP. Theinitial randomized RNA library thus produced comprised 3×10¹⁴ molecules(535 picomoles).

[0060] Eight rounds of SELEX were performed using this randomized RNAlibrary. For each round, Lumino plates (Labsystems, Needham Heights,Mass.) were coated for 2 hours at room temperature with 200 μlDulbecco's PBS containing CIP concentrations as shown in Table 1. Aftercoating, wells were blocked using Superblock Blocking buffer in TBS(Pierce Chemical company, Rockford, Ill.) for rounds 1 to 3 while forrounds 5 to 8 wells were blocked with SHMCK+ buffer [20 mM Hepes, pH7.35, 120 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂ and 1 g/liter casein(I-block; Tropix)]. Binding and wash buffer consisted of SHMCK+ buffercontaining 0.05% Tween 20. For each SELEX round, RNA was diluted into200 μl of binding buffer and allowed to incubate for 2 hours at 37° C.in the protein coated wells that were pre-washed with binding buffer.RNA input into each round is shown in Table 1. After binding, six washesof 200 μl each were performed. Following the wash step, the dry well wasplaced on top of a 95° C. heat block for 5 minutes. Standard AMV reversetranscriptase reactions (50 μl) were performed at 48° C. directly in thewell and the reaction products utilized for standard PCR andtranscription reactions. Two synthetic primers 5N8 (see above) and 3N8:5′-gcctgttgtga gcctcctgtcgaa-3′ (SEQ ID NO:46) were used for thesetemplate amplification and reverse transcription steps. For rounds sixthrough eight, transcriptions were performed in the presence ofbiotin-GAP in order to biotinylate the 5′-termini of the RNA pool.

[0061] The amplified affinity enriched round 8 pool was purified on an8% polyacrylamide gel, reverse transcribed into ssDNA and the DNAamplified by the polymerase chain reaction (PCR) using primerscontaining BamHI and HindIII restriction endonuclease sites. PCRfragments were cloned, plasmids prepared and sequence analyses performedaccording to standard techniques (Sambrook et al. (1989) MolecularCloning: A Laboratory Manual, 2^(nd) Ed. 3 vols., Cold Spring HarborLaboratory Press, Cold Spring Harbor). The sequences of 43 individualclones are shown in FIGS. 1A-1D. The majority of these clones (39)represent one of two distinct sequences designated group A and group B(FIGS. 1A-1C). Sequences obtained from twelve clones were members ofgroup A while sequences obtained from 27 clones were members of group B.Examination of the variable region of the 12 clones in group A revealed9 unique, although highly similar, sequences. Examination of thevariable region of the 27 clones in group B revealed only 7 uniquesequences and these do not differ by more than a few nucleotides (FIGS.1A-1C). In fact, 21 of the sequences are identical and can berepresented by clone DD-172-2-20 (SEQ ID No:13) shown at the top of thegroup B section of FIG. 1A. Finally four individual sequences wereobtained and placed arbitrarily into group C (FIG. 1D). Overall, thisdata represents a highly enriched sequence pool.

Example 2 Affinity Determinations of Affinity Enriched Pool of NucleicAcid Ligands and of Individual Calf Intestinal Phosphatase Nucleic AcidLigands

[0062] The affinity of the initial random library and round 8 affinityenriched pool of Example 1 was performed by a plate-based assay usingRNA-transcripts biotinylated on the 5′-terminus. Briefly, opaque white96-well Lumino plates were coated with streptavidin (10 to 20 μg/well).Biotinylated RNA obtained either from the round 8 pool or the randomlibrary were added separately to individual wells of a 96-wellmicrotiter plate (approximately 30 fMols RNA per well). After incubatingfor 1 hour at room temperature, wells were washed three times with 200ml of SHMCK+ buffer. Varying concentrations of CIP, in SHMCK+ buffer,were added to the wells and allowed to incubate for 1 hour at 37° C.Wells were washed four times with 200 μl SHMCK+ buffer followed by theaddition of a 100 μl solution consisting of 0.1 M diethanolamine pH 10,10% volume/volume Sapphire enhancer solution (Tropix Inc., Bedford,Mass.), 17 ml/ml CSPD (Tropix, Inc.), 1 mM MgCl₂, and 0.02% sodiumazide. After a 30 minute incubation, chemiluminescence was measuredusing a Berthold (Nashua, N.H.) LB 96P luminometer. Luminescence levelswere integrated over one second and data were fit to a sigmoidaldose-response curve (variable slope),Y=Bottom+[(Top−Bottom)/1+10^((Log EC50−X)(Hillslope))] using GraphPadPrism version 2.01 (GraphPad Software; San Diego, Calif.). Eachreplicate was considered individually and convergence was obtained whentwo consecutive iterations varied the sum of squares (relative distanceof the points from the curve, 1/Y2) by less than 0.01%.

[0063] Using this procedure, the affinity-enriched round 8 pool had anequilibrium dissociation constant (Kd) for CIP at 37° C. of 250×10⁻¹² M(FIG. 2; RLU is relative luminescence units). Two representatives ofgroup B, DD-172-2-20 and DD-172-2-4, were tested and displayedsubnanomolar dissociation constants (260 pM and 130 pM respectively).Individual members of group A (DD-172-2-9, DD-172-2-17) and the onemember of group C (DD-172-2-6) were also tested and displayed lownanomolar dissociation constants. (Table 2 and FIG. 3).

Example 3 Measurement of Inhibitory Activity of Calf IntestinalPhosphatase Nucleic Acid Ligands

[0064] CIP was diluted into SHMCK+ buffer (without detergent) to aconcentration of 1 nM. This solution was divided into 250 μl aliquotsand a separate nucleic acid ligand from Example 2 or random RNA-libraryadded to an individual aliquot such that the final aptamer concentrationwas 100 nM. The volumes of nucleic acid ligand addition, ranged from 1.3to 3.6 μl and thus did not significantly alter the concentration of CIP.As a control, one aliquot received 2.5 μl of water. Solutions wereallowed to incubate for 10 minutes at room temperature followed by thetransfer of two 100 μl aliquots, from each original 250 μl aliquot, toduplicate wells of a microtiter plate. After an additional five minuteincubation, 100 μl of 43.6 mM p-Nitrophenyl Phosphate chromagenicsubstrate (pNPP; Sigma, Saint Louis, Mo.) in SHMCK buffer (no detergentand no protein) was added. After 45 minutes the absorbance at 405 nm wasdetermined using a microplate reader. Final absorbance values werecalculated by subtracting the background absorbance as determined fromduplicate wells containing 200 μl of the identical solution exceptwithout the presence of CIP.

[0065] As shown in FIG. 4, under the conditions of this assay neither a2′-F pyrimidine random RNA-library (R) or group 2 clones DD-172-2-4 (4)and DD-172-2-20 (20) were able to inhibit the activity of CIP. However,clones obtained from group 1, namely DD-172-2-9 (9), DD-172-2-17 (17),and DD-172-2-29 (29), reduced the observed CIP activity by 40% despitethe fact that the substrate concentration (21.8 mM) was over 400,000times greater than the aptamer concentration (50 nM). Of course the Kmof CIP for pNPP has not been determined for the buffer conditionsutilized here. TABLE 1 SELEX RNA and protein input. CIP RNA Round(pMol/well) (pMol/well) 1 95 535 2 38 240 3 38 80 4 38 60 5 38 50 6 3850 7 38 43 8 38 20

[0066] TABLE 2 Equilibrium dissociation constants for individual 2′-Fpyrimidine RNA-aptamers at 37 ° C. Clone Kd (pM) 172-2-4 130  172-2-20260  172-2-17 7200 172-2-9 7800 172-2-6 7900

[0067]

1 46 1 87 RNA Artificial Sequence modified_base (1)..(87) Allpyrimidines are 2′F. 1 gggagacaag aauaaacgcu caaacauaaa acaaaauaaccuuagccucg gugcucuacg 60 caauucgaca ggaggcucac aucaggc 87 2 85 RNAArtificial Sequence modified_base (1)..(85) All pyrimidines are 2′F. 2gggagacaag aauaacgcuc aaacacaaaa caaaauaacu uggccucggu gcucuacgca 60auucgacagg aggcucacaa caggc 85 3 88 RNA Artificial Sequencemodified_base (1)..(88) All pyrimidines are 2′F. 3 gggagacaag aauaaacgcucaaacacaaa acaaaaauaa ccuuagccuc ggugcucuac 60 gcaauucgac aggaggcucacaacaggc 88 4 86 RNA Artificial Sequence modified_base (1)..(86) Allpyrimidines are 2′F. 4 gggagacaag aaaaacgcuc aaacacaaaa caaaauaaccuuagccucgg ugcucuacgc 60 aauucgacag gaggcucaca acaggc 86 5 88 RNAArtificial Sequence modified_base (1)..(88) All pyrimidines are 2′F. 5gggagacaag aauaaacgcu caaacacaaa acaaaauaac cuuagccucg gugcucuacg 60caauucgaca ggaggcucac aacaggcg 88 6 87 RNA Artificial Sequencemodified_base (1)..(87) All pyrimidines are 2′F. 6 gggagacaag aauaaacgcucaaacacaaa acaagauaac cuuagccucg gugcucuacg 60 caauucgaca ggaggcucacaacaggc 87 7 86 RNA Artificial Sequence modified_base (1)..(86) Allpyrimidines are 2′F. 7 gggagacaag aauaaacgcu caaacacaaa acaaaaaaccuuagccucgg ugcucuacgc 60 aauucgacag gaggaucaca acaggc 86 8 86 RNAArtificial Sequence modified_base (1)..(86) All pyrimidines are 2′F. 8gggagacaag aauaaacgcu caacacaaaa caaaauaacc uuagccucgg ugcucuacgc 60aauucgacag gaggcucaca acaggc 86 9 86 RNA Artificial Sequencemodified_base (1)..(86) All pyrimidines are 2′F. 9 gggagacaag aauaaacgcucaacacaaaa caaaauaacc uuagccucgg ugcucuacgc 60 aauucgacag gaggcucacaacaggc 86 10 86 RNA Artificial Sequence modified_base (1)..(86) Allpyrimidines are 2′F. 10 gggagacaag aauaaacgcu caacacaaaa caaaauaaccuuagccucgg ugcucuacgc 60 aauucgacag aaggcucaca acaggc 86 11 85 RNAArtificial Sequence modified_base (1)..(85) All pyrimidines are 2′F. 11gggagacaag aauaaacgcu caaacaaaac aaaauaaccc uagccucggu gcucuacgca 60auucgacagg aggcucacaa caggc 85 12 86 RNA Artificial Sequencemodified_base (1)..(86) All pyrimidines are 2′F. 12 gggagacaagaauaaacgcu caaacaaaaa caaaaaauac uuagccucgg ugcucuacgc 60 aauucgacaggaggcucaca acaggc 86 13 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 13 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggcucaca acaggc 86 1486 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 14 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 15 86 RNA ArtificialSequence modified_base (1)..(86) All pyrimidines are 2′F. 15 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 16 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 16 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacug gaggcucaca acaggc 86 1786 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 17 nggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 18 86 RNA ArtificialSequence modified_base (1)..(86) All pyrimidines are 2′F. 18 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 19 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 19 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggcucaca acaggc 86 2086 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 20 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca ucaggc 86 21 86 RNA ArtificialSequence modified_base (1)..(86) All pyrimidines are 2′F. 21 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 22 84 RNA Artificial Sequence modified_base(1)..(84) All pyrimidines are 2′F. 22 gagacaagaa uaaacgcuca acgccaggcccuuaucaagc gcggaacgca ugacccgucu 60 uucgacagga ggcucacaac aggc 84 23 85RNA Artificial Sequence modified_base (1)..(85) All pyrimidines are 2′F.23 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60cuuucgacag gaggcucaca acagc 85 24 85 RNA Artificial Sequencemodified_base (1)..(85) All pyrimidines are 2′F. 24 gggagacaagauaaacgcuc aacgccaggc ccuuaucaag cgcggaacgc augacccguc 60 uuucgacaggaggcucacaa caggc 85 25 88 RNA Artificial Sequence modified_base(1)..(88) All pyrimidines are 2′F. 25 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggggcuca caacaggc 88 2686 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 26 gggagacaag auuaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 27 86 RNA ArtificialSequence modified_base (1)..(86) All pyrimidines are 2′F. 27 gggagacaagaaucaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 28 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 28 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggcucaca acaggc 86 2986 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 29 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 30 86 RNA ArtificialSequence modified_base (1)..(86) All pyrimidines are 2′F. 30 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 31 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 31 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggcucaca acaggc 86 3286 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 32 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 33 85 RNA ArtificialSequence modified_base (1)..(85) All pyrimidines are 2′F. 33 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcgcggaacg caugacccgu 60 cuucgacaggaggcucacaa caggc 85 34 85 RNA Artificial Sequence modified_base(1)..(85) All pyrimidines are 2′F. 34 gggagacaag aauaaacgcu caacgccaggccccuaucaa gcgcggaacg caugacccgu 60 cuuucgacag gaggcucaca acagc 85 35 86RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are 2′F.35 gggagacaag aauaaacgcu caacgccagg cccuuaucaa gcgcggaacg cacgacccgu 60cuuucgacag gaggcucaca acaggc 86 36 86 RNA Artificial Sequencemodified_base (1)..(86) All pyrimidines are 2′F. 36 gggagacaagaauaaacgcu caacgccagg cccuuaucaa gcguggaacg caugacccgu 60 cuuucgacaggaggcucaca acaggc 86 37 86 RNA Artificial Sequence modified_base(1)..(86) All pyrimidines are 2′F. 37 gggagacaag aauaaacgcu caacgccaggcccuuaucaa gcgcggaacg caugacccgu 60 cauucgacag gaggcucaca acaggc 86 3886 RNA Artificial Sequence modified_base (1)..(86) All pyrimidines are2′F. 38 gggagacaag aauaaacgcu caaugccagg cccuuaucaa gcgcggaacgcaugacccgu 60 cuuucgacag gaggcucaca acaggc 86 39 85 RNA ArtificialSequence modified_base (1)..(85) All pyrimidines are 2′F. 39 gggagacaagaauaaacgcu caagccaggc ccuuaucaag cgcggaacgc augacccguc 60 uuucgacaggaggcucacaa caggc 85 40 87 RNA Artificial Sequence modified_base(1)..(87) All pyrimidines are 2′F. 40 gggagacaag aauaaacgcu caaguaaaagccucauagaa cuaacuaaca acgcgcucac 60 ggauucgaca ggaggcucac aacaggc 87 4185 RNA Artificial Sequence modified_base (1)..(85) All pyrimidines are2′F. 41 gggagacaag aauaaacgcu caacgucaua aauguaaaca ggauuauaagcgcgaccuag 60 auucgacagg aggcucacaa caggc 85 42 81 RNA ArtificialSequence modified_base (1)..(81) All pyrimidines are 2′F. 42 gggagacaagaauaaacgcu caagcgcaaa gccuuucaag cucgugcuau cacugaauuc 60 gacaggaggcucacaacagg c 81 43 87 RNA Artificial Sequence modified_base (1)..(87)All pyrimidines are 2′F. 43 gggagacaag aauaaacgcu caacgucccc ccgucaagaucuccucccuc cgcguccccu 60 cccuucgaca ggaggcucac aacaggc 87 44 87 DNAArtificial Sequence Description of Artificial Sequence SyntheticSequence 44 gcctgttgtg agcctcctgt cgaannnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn 60 nnnnttgagc gtttattctt gtctccc 87 45 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 45taatacgact cactataggg agacaagaat aaacgctcaa 40 46 24 DNA ArtificialSequence Description of Artificial Sequence Synthetic Sequence 46gcctgttgtg agcctcctgt cgaa 24

What is claimed is:
 1. A reagent for the detection of a target molecule,the reagent comprising: a) a nucleic acid ligand to said target; and b)a nucleic acid ligand to calf intestinal phosphatase (CIP); wherein thesequence of said nucleic acid ligand to said target is contiguous withthe sequence of said nucleic acid ligand to CIP.
 2. The reagent of claim1 wherein said nucleic acid ligand to CIP is a non-inhibitory ligand. 3.The reagent of claim 1 wherein said nucleic acid ligand to CIP is aninhibitory ligand.
 4. The reagent of claim 3 wherein the binding of saidtarget to said nucleic acid ligand to said target releases any CIP thatis bound to said nucleic acid ligand to CIP.
 5. A method for determiningthe quantity of a target molecule in a sample suspected of containingsuch target molecule, the method comprising: a) providing a reagent forthe detection of said target molecule, the reagent comprising: i) anucleic acid ligand to said target; and ii) an inhibitory nucleic acidligand to calf intestinal phosphatase (CIP); wherein the sequence ofsaid nucleic acid ligand to said target is contiguous with the sequenceof said nucleic acid ligand to CIP, and wherein the binding of saidtarget to said nucleic acid ligand to said target releases any CIP thatis bound to said inhibitory nucleic acid to CIP; b) contacting saidreagent with a predetermined quantity of CIP, wherein substantially allof the CIP is bound and inhibited by said inhibitory nucleic acid ligandto CIP; c) contacting said reagent and said predetermined quantity ofCIP with said sample to form a test mixture; and d) measuring the CIPactivity in said test mixture; whereby binding of said target in saidsample releases CIP that is bound to the inhibitory nucleic acid ligandto CIP of said reagent.
 6. The method of claim 5 wherein step d) isaccomplished using a detectable substrate of calf intestinalphosphatase.
 7. The method of claim 6 wherein said detectable substrateis selecting from the group consisting of 1,2 dioxetane andp-nitrophenylphosphate.
 8. An antibody conjugated to a non-inhibitorynucleic acid ligand to calf intestinal phosphatase (CIP).
 9. A method ofdetermining the quantity of a target molecule in a sample suspected ofcontaining said target molecule, the method comprising: a) immobilizingsaid sample on a solid support; b) contacting said immobilized samplewith an antibody to said target, wherein said antibody is conjugated toa non-inhibitory nucleic acid ligand to calf intestinal phosphatase(CIP); c) contacting said immobilized sample with CIP; and d)determining the level of CIP activity.
 10. The method of claim 9 whereinstep d) is accomplished using a detectable substrate of calf intestinalphosphatase.
 11. The method of claim 10 wherein said detectablesubstrate is selecting from the group consisting of 1,2 dioxetane andp-nitrophenylphosphate.